1. Field of the Invention
The present invention relates to a method for producing a highly efficient antibody which takes the most important role in immunoassay useful in the fields of medical diagnosis, chemical analysis, etc., and more particularly, to a method for producing an antibody having a high affinity to low molecular weight compound, namely, hapten which is per se incapable of causing immunoresponse.
In more detail, the present invention relates to the production of an antibody or monoclonal antibody having a high affinity to methamphetamine which exerts on a central excitatory action and is one of analeptics.
2. Related Art Statement
In detecting a trace component in blood or a specific component in the atmosphere, it is necessary to detect with certainty a specific component present in a trace amount from very many impurities. In recent years, immunoassay based on the reaction of an antibody with an antigen has been extensively investigated for this purpose.
Immunoassay is roughly classified into radioimmunoassay using a radioactive isotope and enzyme-immunoassay (EIA) using an enzyme. From viewpoints of safety and simplicity, EIA is more advantageous.
Enzyme-linked immunosorbent assay (ELISA) which is typical EIA is described below by referring to FIG. 1. In FIG. 1, numeral 1 denotes a microplate made of plastic such as polystyrene which non-specifically adsorbs protein. Numeral 2 denotes a solid phase antigen adsorbed onto microplate 1, which antigen is prepared by introducing a suitable functional group into an antigen as analyte and chemically binding the antigen to protein via the functional group. Numeral 3 is an antibody capable of binding to analyte and the antibody binds to solid phase antigen 2 in an equilibrated state. That is, antibody 3 is immobilized to microplate 1 via solid phase antigen 2. When an antigen 4 as analyte is introduced into the system, solid phase antigen 2 and antigen 4 as analyte competitively bind to antibody 3. Therefore, a part of antibody 3 binds to antigen 4 as analyte and as the consequence, a part of antibody 3 remains unbound to microplate 1. As a matter of course, the larger the quantity of antigen 4 as analyte, the more antibody 3 unbound to microplate 1 increases. At this stage, the unbound antibody 3 can be removed by washing. After removing the unbound antibody 3, antibody 5 capable of binding to antibody 3 is added to the system. Antibody 5 chemically binds to enzyme 6 in a definite proportion. After antibody 5 unbound to antibody 3 is removed by washing, the activity of enzyme 6 is determined. There is a positive correlation between the activity and the quantity of enzyme 6 so that the quantity of enzyme 6 can be quantitatively determined by assaying the reactivity. When the quantity of enzyme 6 is known, it is possible to quantitatively determine the quantity of antibody 3 bound to microplate 1 via solid phase antigen 2 and then quantitatively determine antigen 4 as analyte. In this case, a primary factor to determine the detection sensitivity is an affinity of antibody 3 to antigen 4 as analyte.
An antibody is one of protein produced by the organism which has a property of selectively binding only to a specific substance, i.e., antigen. An antigen is roughly classified into two types due to differences in the production of an antibody binding to the antigen. One is an antigen that can induce production of an antibody by injecting the antigen directly into animal. In general, macromolecular substances having a molecular weight of several ten thousands or more correspond to such an antigen. Another has a relatively small molecular weight and, by chemically bonding to a suitable protein, it can acts as an immunogen inducing production of an antibody for the first time. In particular, the latter antigen is called hapten. A method for producing an antibody to hapten as antigen is described in Haptens and Carriers, O. Makela and I. J. T. Seppala, Handbook of Experimental Immunology, 4th edition, Volume 1, Chapter 3, edited by D. M. Weir, Blackwell Scientific Publications, Oxford, 1986. That is, it is described that where hapten contains a functional group capable of chemical binding, hapten is bound to protein via the functional group to form an immunogen. If any functional group is absent in the hapten, an immunogen is generally obtained by introducing an appropriate functional group such as an amino group, a carboxyl group and a hydroxyl group and a spacer composed of an alkyl group having 1 to about 10 carbon atoms into the hapten to synthesize hapten derivatives and binding the derivatives to a suitable protein using a crosslinking agent. More specifically, Takami et al. reports the production of antibody using as a hapten methamphetamine (MA) having a central excitatory action which is one of analeptics [Takami, Fukuda and Takahashi, Japan, J. Legal Med., 37 (4), 417, 1983]. That is, it is reported that aminobutyl group is introduced to the secondary amino group of MA to produce aminobutyl methamphetamine (ABMA). The derivative is bound to protein and the obtained product is used as an immunogen.
However, the present inventors' study has revealed that the antibody produced using the immunogen has a higher affinity to the antigen derivative, i.e., ABMA, rather than to the desired antigen, i.e., MA. That is, the antibodies produced by the prior art methods are antibodies not to the antigen but to the antigen derivatives. The antigen derivatives have a structure different from that of the antigen, because functional groups, spacers, etc. are introduced. Thus, there is clearly revealed a problem that an antibody having a high affinity to the desired antigen is produced only with difficulty As the result, upon assaying an antigen as analyte according to, for example, ELISA described above, using the antibody produced by the prior art methods, even though the antigen an analyte is introduced into the system in large quantities, the unbound antibody does not increase, so that it is impossible to detect the antigen as analyte with high sensitivity.